Detecting and mapping of inflamed zones in a living tissue

ABSTRACT

A method for detecting and mapping inflamed zones in living tissues and a device for implementing the method. The method includes subjecting the tissues to be analyzed to a luminous excitation with a predetermined spectral domain, acquiring at least the raw fluorescence signal of the porphyrins for a plurality of measurement points, and in determining, for each measurement point, the intensity of the fluorescence for the wavelengths characteristic of endogenous porphyrins.

CROSS REFERENCE TO RELATED APPLICATION

This is the 35 USC 371 national stage of International ApplicationPCT/FR98/01950 filed on Sep. 11, 1998 which designated the United Statesof America.

FIELD OF THE INVENTION

The present invention relates to the field of examination and analysisof living tissues, and has for its object a process for the detectionand mapping of inflamed zones of living tissues, as well as a device topractice the same.

BACKGROUND OF THE INVENTION

The term “inflammation” generally designates, and particularly in thepresent case, all the processes developed by living organisms inresponse to an aggression of internal or external origin.

Inflammation gives rise to the major cellular histocompatibility systemcomprising leukocytes, lymphocytes, monocytes, histocytes, macrophages,etc . . . , as well as their secretion products such as prostaglandins,histamines, serotonins and cytokines (interferons, interleukins, tumoralnecrosis factors . . . ).

It is already known to use the fluorescence of an exogensis chromophoreof the type derived from hematoporphyrin or of the precursor type ofprotoporphyrine IX (5-amino-levulinic acid) to detect inflammatory orcancerous lesions.

Thus, it has been shown that these exogenous chromophores, concentrate,after injection, more particularly in the inflammatory or cancerouslesions.

Moreover, there are also known methods for the detection of cancersbased on emission of autofluorescence in the spectral band of bluelight.

In these latter methods, one takes account of the endogenouschromophores derived from nicotinamides (NAD, NADH), or constituting anextracellular matrix such as collagens, elastins or flavin derivatives.

Moreover, the presence of porphyrins in tissues has been known since1920. It has been observed in tumors, and then in the Harder gland(located behind the eye muscles) of rodents and finally in very smallquantity in normal tissues. It is principally protoporphyrin IXsynthesized in cells from 5-amino-levulinic acid, that is the first stepof the synthesis of hemoglobin.

The reason for the presence of this enzymatic path (normally expressedin the bone marrow) in normal or tumorous tissues, remains for themoment unknown, although it has recently been shown that this synthesisis under the control of certain hypophysary hormones.

SUMMARY OF THE INVENTION

However, the inventors of the present invention have discovered, in anunexpected and surprising manner, the presence of an abnormally highquantity of porphyrins in numerous situations whose common character isthe existence of an inflammation. Present at very low levels in healthytissues which have been studied (muscles, esophagus, pancreas and liver,at a slightly higher level in this latter, the site of partialdestruction of the red corpuscles), these endogenous porphyrins becomevery abundant in these same tissues after aggression by a trauma(muscle), an irritating product (liver), by the development of a cancer(esophagus, pancreas) or an acute or chronic experimental pancreatitis.

The present invention has for its present object a process for thedetection and mapping of inflamed zones of living tissues, characterizedin that it consists in subjecting the tissues to be analyzed to aluminous excitation in a predetermined spectral field, acquiring atleast the unprocessed fluorescence signal of the porphyrins for aplurality of points of measurement, and determining, for each point ofmeasurement, at least the intensity of fluorescence for wavelengthscharacteristic of endogenous porphyrins.

The invention also has for its object a device for practicing thementioned process, principally constituted by a luminous excitation unitof low intensity in the spectral bands centered about 400 nm and about590 nm, a filtering module comprising a set of pass-band filters adaptedto select fluorescenses specific to the different chromophores inquestion, a unit for detection and recordation of images of thefluorescence of the surface of the examined tissues and, finally, a dataunit for processing point by point, or pixel by pixel, of the imagesrecovered and the command and control of the assembly of the device,associated with storage and edition means for images.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood from the following description,which relates to a preferred embodiment, given by way of non-limitingexample, and explained with reference to the accompanying schematicdrawings, in which:

FIG. 1 shows the fluorescence spectra, on the one hand, of muscles inthe course of healing (during the so-called “inflammatory” phase) and,on the other hand, of an undamaged muscle (broken line);

FIG. 2 shows the fluorescence spectra, on the one hand, of a liverinjured by retrograde injection of taurocholate (to reproduce a hepatictype lesion) and, on the other hand, of an undamaged liver (in brokenline);

FIG. 3 shows the fluorescence spectra, on the one hand, of esophagi withadenocarcinomas, and, on the other hand, an undamaged esophagus (in finebroken line);

FIG. 4 shows the fluorescence spectra, on the one hand, of a pancreashaving increasing stages of acute pancreatic necrotico-hemorrhage, and,on the other hand, an undamaged pancreas (in broken line);

FIG. 5 shows the fluorescence spectra, on the one hand, of pancreaseshaving adenocarcinomens of the ductal phenotype and, on the other hand,of an undamaged pancreas (in broken line);

FIG. 6 shows the fluorescence spectra, on the one hand, of healthyregions of a pancreas having adenocarcinomas and, on the other hand, anundamaged pancreas (in broken line);

FIG. 7 shows the fluorescence spectra, on the one hand, of a pancreashaving chronic pancreatitis and, on the other hand, a healthy pancreas(in broken line);

FIGS. 8 and 9 show fluorescence images of a rat pancreas having acutepancreatitis, taken at 630 nm, at 680 nm (replacing the ideal value of600 nm) and at 470 nm, the images obtained after processing bysubtraction and by division point by point according to the process ofthe invention and the final images used for location (markedPhoto+Processed), and,

FIG. 10 is a schematic view of a device for practicing the processdescribed above.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the process for the detection and mapping ofinflamed zones of living tissues, consists essentially in subjecting thetissues to be analyzed to a luminous excitation with a predeterminedspectral range, acquiring at least the unprocessed fluorescence signalof porphyrins for a plurality of measuring points, and determining, foreach measurement point, the intensity of fluorescence for thewavelengths characteristic of endogenous porphyrins.

By endogenous porphyrins, the inventors mean porphyrins normally presentin tissue without any exogenous addition, particularly without theaddition of an external agent giving rise to an increase in theformation of porphyrins, such as for example δ-aminolevulinic acid (ALA)precursor of protoporphyrin IX.

FIGS. 1 to 7 show examples of the fluorescent spectra of differenthealthy tissues and of the same tissues having inflammations of variousorigins.

It should be noted that there can be seen, in all these spectra, afluorescent emission band in the red (substantially comprised between600 and 800 nm) of more or less high intensity and which systematicallyhas two emission peaks that are easily recognizable, namely a principalpeak at 630 nm and a secondary peak at 690 nm, this double peaking beingcharacteristic of porphyrin fluorescence.

It will also be seen from these figures that in all the cases ofinflammation shown, a maximum of the fluorescence band in the blue hasan offset toward the green of about 50 nm.

Moreover, there has also been noticed, independently of the absolutevalue of fluorescence (very difficult to estimate for dense tissues), anincrease of the ratio of the intensities of fluorescence emitted in thered (porphyrins) to those emitted in the blue (constituting the cellularmatrix and extra cellular matrix), relative to a corresponding increaseof the seriousness of inflammation.

This dependent relation indicates the existence of a direct correlationbetween, on the one hand, the concentration of porphyrins, and hence thevalue of the intensity of fluorescence for wavelengths characteristic ofporphyrin and, on the other hand, the importance of the intensity of theinflamed lesions, permitting the gradation of inflammatory character ofa tissue lesion.

This possibility of carrying out a gradation of the gravity of theinflammatory process has been verified by the inventors by comparisonbetween the macroscopic phenomena observed in the autopsy of animalshaving pancreatitis and the measurements of fluorescence of theendogenous porphyrins carried out on the corresponding pancreases.

It will thus be seen that in the case of reduced pancreatitis (simpleedema of the gland) this fluorescence is multiplied by two, incomparison to that of the normal or healthy pancreatic tissue. Severepancreatitis, leading to death of the animal, with the autopsy of anecrotic pancreatic gland, is correlated to a fluorescence of theendogenous porphyrins five times higher than in the case of simpleedemic pancreatitis.

FIG. 4 shows the corresponding spectra for these clinicalsituations—namely healthy pancreas, simple edemic pancreatitis,necrotico-hemorrhagic pancreatitis—with moreover the spectrum of anintermediate pancreatitis.

According to a preferred embodiment of the invention, the processconsists in forming a fluorescent image of the tissues to be analyzed,on which appears the inflamed zone or zones, by using at least thepredetermined number of the florescent intensity characteristic of theendogenous porphyrins for different measurement points in question.

Thus, the process according to the invention permits particularlyappreciating, by measuring the fluorescence of the porphyrins, theinflammatory character of an epithelium (skin and mucosa), andestablishing a predictive factor of its ultimate development (forexample a cheloid scar or the malignant transformation of a pigmentedmole).

It also permits detecting inflammations of the epithelia of thepulmonary, digestive, urinary or genital passages by endoscopicmeasurement of the fluorescence of the porphyrins and establishing agradation factor of the lesions (for example the esophagus of Barret:inflamed lesion of the esophagus developing progressively to cancer) orfollowing their development (for example, peptic ulcer of the stomachwhich can develop into healing, a fibrous scar or cancer).

It also permits determining, for example, during the course of asurgical intervention or of a CPRE (choledocho-pancreatic retrogradeendoscopy), the level of information of acute or chronic pancreatitis bymeasurement of the fluorescence of porphyrins in the pancreatic gland orthe excretory passages.

In this latter case, the predictive character in terms of gravity ofpancreatitis, of the results flowing from the process according to theinvention, could be added to the present medical prognostic criteriasuch as the Ranson score (graduated intensity from 3 to 11) or thepancreatic tomodensitometry (stages D and E).

According to one characteristic of the invention, the luminousexcitation is of low intensity, with a power density of delivered energypreferably at most equal to 0.5 W/cm² (so as not to give rise to localtemperature elevation of more than 1° C.).

Moreover, said luminous excitation is preferably constituted by twowavelengths or two spectral bands, namely one of about 590 nm orcentered on 590 nm and adapted to excite the porphyrins, and the otherof about 400 nm or centered on 400 nm (or if desired about 355 nm),adapted to excite other endogenous chromophores.

The process according to the invention consists more particularly, afterthe excitation phase, in acquiring, for the same tissues to be analyzed,fluorescence signals in the spectral bands centered, respectively, onabout 600 nm (or else about 680 nm) and on about 630 nm and/or 680-690nm, and, as the case may be, on about 470 nm and/or 510-520 nm, for eachof the points to be measured, to form for each above-mentioned spectralband, a fluorescence image from values of fluorescence intensity takenin the spectral band in question for the different points ofmeasurement, carrying out a normalization of the mean values of theintensities of each image obtained, and processing point by point theimage or the images obtained for the spectral band or bands centered onabout 630 nm and/or about 680-690 nm and using the data contained in thenormalized image obtained, for the pass-band centered on 600 nm and, asthe case may be, supplemental processing by using the data contained inthe image or images obtained for the spectral band or bands centered onabout 470 nm and/or about 510-520 nm.

In the frequency bands indicated above, it should be noted that: 470 nmcorresponds to the spectral position of the peak of autofluorescence inhealthy tissues; 510 nm corresponds to the spectral position of the peakof blue autofluorescence in inflamed tissues; 600 nm (associated with anexcitation at 510 nm) corresponds to the autofluorescence in the redspectral band considered as the baseline (background noise) forfluorescence of porphyrins; 630 nm and 690 nm correspond to the spectralpositions of the major and minor emission peaks of endogenousporphyrins.

According to one characteristic of the invention, it can preferably beprovided to use a normalization factor of a given value for the imagescollected at 470 nm and/or at 510 nm and another normalization factor ofa different value for the images collected at 600 nm and 630 nm and/or690 nm, the values of these two factors being defined so as to obtain anoptimum gradation of the colors or the levels of gray on the final imageafter processing.

So as to isolate the fluorescence signal generated by the porphyrins,there is carried out a point by point subtraction, or pixel by pixelsubstraction, of the images of the intensities of the normalized imagecollected at 600 nm, from those of the normalized image or imagescollected at 630 and/690 nm.

Moreover, so as to avoid problems of collection of the fluorescence due,among other things, to tissue heterogeneity, one can proceed, aftersubstraction of the normalized image intensities collected at 600 nm, toa point by point division, or pixel by pixel division, of the imageintensities or normalized images collected at 630 and/or 690 nm, by thenormalized image intensities collected at 470 nm or 510 nm.

As a modification, one can proceed, after subtraction of the intensitiesof the normalized image collected at 600 nm, to a point by point or apixel by pixel division of the intensities of the normalized image orimages collected at 630 and/or 690 nm by the intensities of the imageobtained by point by point substraction of the intensities of thenormalized image collected at 510 nm, from those of the normalized imagecollected at 470 nm, this manner of proceeding improving the contrastbetween healthy tissues and inflamed tissues.

Thus, according to one preferred embodiment, the operations of imageprocessing can consist more particularly in making the pixel by pixelratio between the fluorescence image of the endogenous porphyrins(I₆₃₀+I₆₈₀, after subtraction of the “background noise” taken at 600 nm)and that of the fluorescence of the NADPH (I₄₇₀), which is more intensein the healthy tissues than in the sick tissues.

So as to improve, if necessary, the contrast between the differentconditions of the tissue, the process can also use an increase ofautofluorescence to 510-520 nm in the process of inflammatory processes,which increase is disclosed by the inventors. In this case, amultiplication of the image ratio [(I₆₃₀+I₆₈₀−background noise)/I₄₇₀] bythe image collected at 510-520 nm (I₅₁₀₋₅₂₀) is predetermined.

The object of normalizing the images recorded at the differentwavelengths (470, 510-520, 630 and 680, after substraction of noise) isto increase the content of the pixels of the final image resulting fromthe process described above, so as to increase the dynamic of the finalimage and thereby to have better localization and visualization of theinflamed portion.

Thanks to the arrangements for image processing mentioned above, theprocess according to the invention permits obtaining a final image onwhich is visible only the inflamed zone or zones.

FIGS. 8 and 9 show two examples of localization of zones of inflamedlesions due to acute pancreatitis (weak or moderate) in rat, by the useof a process according to the invention.

These figures therefore show images of fluorescence of theduodenal-pancreatic region, collected by a CCD digital camera. Theseimages have been obtained with laser excitation through pass-bandfilters permitting selecting fluorescent emissions in the spectralregions characteristic of porphyrins (I_(630 nm)), of autofluorescencein the red (I_(680 nm)) and of autofluorescence in the blue(I_(470 nm)).

The operations of processing images according to the invention permitobtaining first of all the image of the fluorescence of porphyrinsproperly so-called (I_(630 nm-680 nm)), then eliminating the contingentoutput of fluorescence by dividing this latter image by that of the blueautofluroescence (obtained from the marked imageI_(630 nm-680 nm/470 nm)).

The superposition of this latter image on a normal photographic imagepermits precisely locating the inflammation zone (Photo+Processed).

The invention also has for its object a device for practicing theprocess described above, shown in FIG. 10 of the accompanying drawings.

This device is principally constituted by a luminous excitation unit 1of low intensity in the spectral bands centered on about 400 nm andabout 590 nm, a filtering module 2 comprising a set of pass-band filtersadapted for selecting specific fluorescenses at the different soughtchromophores, a unit 3 for detecting and recording images of thefluorescence of the surface of the tissues examined and, finally, acomputing unit 4 for the point by point or pixel by pixel processing ofthe images collected and the command and management of the assembly ofthe device, associated with storage and editing means for the images(not shown).

The luminous excitation unit 1 could be comprised either of lasers ofsuitable wavelength, or of lamps provided with pass-band filterscentered on the wavelengths of the mentioned excitation.

Preferably, the unit 3 for detecting and recording images, in falsecolors, consists of a digital camera CCD.

According to one characteristic of the invention, the excitation unit,the filtering module and the unit for detecting and recording images,are preferably adapted to coact with the optical means used duringendoscopy.

Under conditions in which the fluorescent signals will not have muchintensity (low excitation or output of fluorescence), it can be providedto add to the CCD camera an image intensifier 5 with a variable gain(typically up to 10³).

For use under standard clinical conditions, which is to say in ambientlight, this intensifier 5 will be preferentially closable and used inconnection with a pulstolated laser 1. Opening the intensifier should besynchronized with the pulses of the excitation laser and the duration ofopening less than 100 ns. This will have the advantage on the one handof avoiding possible saturation of the camera by the ambient light andon the other hand to improve the signal/noise ratio.

If the lifetime of the fluorescence of endogenous porphyrins becomessubstantially greater than that of the normal tissue fluorescence, animprovement of the contrast between healthy tissue and inflamed tissuecan be obtained by opening the intensifier with a certain delay relativeto the excitation for recording the fluorescent images of theporphyrins. There will thus be drastically decreased the contribution ofthe fluorescence from molecules other than porphyrins and of which thegreatest portion of the emission would take place before opening theintensifier.

Thanks to the invention, it is therefore possible to carry out detectionand mapping of the inflamed zones of living tissues, as well as anevaluation of the intensity of inflammation, according to a non-invasiveevaluation process and without any addition or drugs or injection ofchromophores.

Moreover, the process according to the invention preserves the integrityof the patient because it is not necessary to carry out any hypodermicwork or removal of tissue, nor to have any direct contact of theapparatus with the tissues to be examined in the course of acquiringdata.

Moreover, the use of this process could be used in making either anexternal examination of the process, or in the scope of the productionof an investigative mode already validated, to which no substanceharmful to the patient will be added.

Of course, the invention is not limited to the embodiment disclosed andshown in the accompanying drawings. Modifications remain possible,particularly as to the construction of the various elements or bysubstitution of technical equivalents, without thereby departing fromthe scope of protection of the invention.

What is claimed is:
 1. Process for the detection and mapping of inflamedzones of living tissues, which comprises: subjecting the tissues to beanalyzed to a luminous excitation with a spectral field; acquiring anunprocessed fluorescent signal of endogenous porphyrins at a pluralityof measurement points; acquiring, for the same tissues to be analyzed,fluorescence signals in the spectral bands centered respectively onabout 600 nm and on at least one of about 630 nm and 680-690 nm, and onat least one of about 470 nm and 510-520 nm, for each of the measurementpoints; forming for each spectral band a fluorescence image from valuesof fluorescence densities taken in the spectral band in question for thedifferent measurement points, using a normalization factor of a givenvalue for images collected at at least one of 470 nm and 510 nm andanother normalization factor of a different value for images collectedat at least one of 600 nm, 630 nm, and 690 nm, the values of these twofactors being defined so as to obtain an optimum gradation of the colorsor levels of gray on the final image after processing; and determining,for each measurement point, the fluorescence intensity for wavelengthscharacteristic of the endogenous porphyrins.
 2. The process according toclaim 1, wherein the tissues to be analyzed are from theduodenal-pancreatic region.
 3. The process according to claim 1, whereinthe fluorescence image or the tissues to be analyzed on which at leastone inflamed zone appears is formed by using values of the fluorescenceintensity suitable for endogenous porphyrins for the differentmeasurement points.
 4. The process according to claim 1, furthercomprising determining a gradation of the inflammatory character of thezones or lesions by correlation with the fluorescence intensity for thewavelengths characteristic of porphyrins.
 5. The process according toclaim 1, characterized in that the luminous excitation has a powerdensity of delivered energy at most equal to 0.5 W/cm², and constitutedof two wavelengths or two spectral bands, namely one about 590 nm orcentered on 590 nm adapted for the excitation of porphyrins and theother about 400 nm or centered on 400 nm, adapted for the excitation ofthe other endogenous chromophores.
 6. The process according to claim 1,further comprising carrying out a normalization of the mean values ofthe intensities of each image obtained and carrying out a point by pointprocessing of the image or images obtained for the spectral band orbands centered on at least one of about 630 nm and about 680-690 nm byusing the data contained in the normalized image obtained for thepass-band centered on 600 nm, and a supplemental processing by usinginformation contained in the image or images obtained for the spectralband or bands centered on at least one of about 470 nm and about 510-520nm.
 7. The process according to claim 6, further comprising carrying outa subtraction point by point, or pixel by pixel, of the normalized imageintensities collected at 600 nm, from those of the normalized image orimages collected at at least one of 630 and 690 nm.
 8. The processaccording to claim 7, further comprising carrying out, after subtractionof the normalized image intensities collected at 600 nm, a point bypoint or pixel by pixel division of the image intensities or of thenormalized images collected at at least one of 630 and 690 nm, withintensities of the normalized image collected at 470 nm or 510 nm. 9.The process according to claim 7, further comprising carrying out, aftersubtraction of the normalized image intensities collected at 600 nm, apoint by point or pixel by pixel division of the image densities or thenormalized images collected at at least one of 630 nm and 690 nm, byintensities of the image obtained by point by point subtraction of theintensities of the normalized image collected at 510 nm from those ofthe normalized image collected at 470 nm.
 10. The process according toclaim 6, further comprising making the ratio, pixel to pixel, betweenthe fluorescence image of the endogenous porphyrins:(I₆₃₀+I₆₈₀−background noise at 600 nm) and that of a NADPH fluorescenceI₄₇₀; and carrying out a multiplication of the obtained image ratio:(I₆₃₀+I₆₈₀−background noise)/I₄₇₀) by the fluorescence image collectedat 510-520 nm.
 11. Device for the detection and mapping of inflamedzones of living tissues, which comprises: a luminous excitation unithaving an intensity equal to at most 0.5 W/cm², in the spectral bandscentered on about 400 nm and on about 590 nm; a filtering modulecomprising a set of pass-band filters adapted for the selection ofphosphorescences specific to endogenous porphyrins; means for acquiringunprocessed fluorescent signal of the endogenous porphyrins at aplurality of measurement points; a unit for detecting and recordingimages of the fluorescence of the surface of the tissues to be analyzed;means for acquiring, for the same tissues to be analyzed, fluorescencesignals in the spectral bands centered respectively on about 600 nm andon at least one of about 630 nm and 680-690 nm, and on at least one ofabout 470 nm and 510-520 nm, for each of the measuring points; means forforming for each spectral band a fluorescence image from values offluorescence densities taken in the spectral band in question for thedifferent measurement points; a data processing unit for the point bypoint or pixel by pixel processing of images collected and the controland management of the assembly of the device, associated with means forstoring and editing the images, to detect and map the inflamed zones ofthe tissues; said data processing unit using a normalization factor of agiven value for images collected at at least one of 470 nm and 510 nmand another normalization factor of a different value for imagescollected at 600 nm and at at least one of 630 nm and 690 nm, the valuesof these two factors being defined so as to obtain an optimum gradationof the colors or levels of gray on the final image after processing; andmeans for determining, for each measurement point, the fluorescenceintensity for wavelengths characteristic of the endogenous porphyrins.12. The device according to claim 11, wherein the unit for detecting andrecording images comprises a digital camera CCD, and the excitationunit, the filtering module and the unit for detecting and recordingimages, are adapted to be operatively associated with optical means usedin endoscopy.
 13. The device according to claim 12, wherein the digitalcamera CCD includes an image intensifier with variable gain.
 14. Thedevice according to claim 12, further comprising means for carrying outa normalization of the mean values of the intensities of each imageobtained and carrying out a point by point processing of the image orimages obtained for the spectral band or bands centered on at least oneof about 630 nm and about 680-690 nm by using the data contained in thenormalized image obtained for the pass-band centered on 600 nm, and asupplemental processing by using information contained in the image orimages obtained for the spectral band or bands centered on at least oneof about 470 nm and about 510-520 nm.
 15. The device according to claim14, wherein the data processing unit carries out a subtraction point bypoint, or pixel by pixel, of the normalized image intensities collectedat 600 nm, from those of the normalized image or images collected at atleast one of 630 and 690 nm.
 16. The device according to claim 15,wherein the data processing unit carries out, after subtraction of thenormalized image intensities collected at 600 nm, a point by point orpixel by pixel division of the image intensities or of the normalizedimages collected at at least one of 630 and 690 nm, with intensities ofthe normalized image collected at 470 nm or 510 nm.
 17. The deviceaccording to claim 15, wherein the data processing unit carries out,after subtraction of the normalized image intensities collected at 600nm, a point by point or pixel by pixel division of the image densitiesor the normalized images collected at at least one of 630 and 690 nm, byintensities of the image obtained by point by point subtraction of theintensities of the normalized image collected at 510 nm from those ofthe normalized image collected at 470 nm.
 18. The device according toclaim 14, wherein the data processing unit effectuates a ratio, pixel topixel, between the fluorescence image of the endogenous porphyrins:(I₆₃₀+I₆₈₀−background noise at 600 nm) and that of a NADPH fluorescenceI₄₇₀, and carries out a multiplication of the obtained image ratio:(I₆₃₀+I₆₈₀−background noise)/I₄₇₀) by the fluorescence image collectedat 510-520 nm.